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Biochemistry 1986, 25, 4322-4329
Purification of a Benzo[ alpyrene Binding Protein by Affinity Chromatography and Photoaffinity Labeling? Sheila Collins: and Michael A. Marietta* Program in Toxicology, Department of Applied Biological Sciences, Massachusetts Institute of Technology, Cambridge, Massachusetts 021 39 Received October 15. 1985; Revised Manuscript Received February 24, 1986
ABSTRACT: Binding proteins for the polycyclic aromatic hydrocarbon carcinogen benzo [a] pyrene (B [alp)
have been purified from C57B 1/6J mouse liver. Following affinity chromatography on aminopyreneSepharose, a single polypeptide of 29 000 daltons was isolated. The photolabile compound 1-azidopyrene was developed as a photoaffinity labeling agent to identify the protein during its purification. 1-Azidopyrene was found to be a competitive inhibitor of [3H]B[a]P binding. Affinity labeling studies with [3H]-1-azidopyrene in unfractionated cytosol, and in purified preparations, yielded a single covalently labeled protein of 29 000 daltons. The formation of this labeled species was blocked by preincubation with excess unlabeled B[a]P. A native molecular weight of 30000 was estimated by gel filtration chromatography of [3H]B[a]Pand [3H]-1-azidopyrene-labeled cytosol proteins. An equilibrium dissociation constant of 2.69 f 0.66 nM and a maximum number of binding sites of 2.07 f 0.10 nmol of [3H]B[a]P bound/mg of protein were estimated for the pure protein. Two-dimensional gel electrophoresis further resolved the purified 29 000-dalton protein into three major isoelectric variants, each of which was specifically labeled by [3H]-1 -azidopyrene.
polycyclic aromatic hydrocarbons are considered to be relatively inert molecules biologically until their metabolic transformation to reactive electrophilic species. The primary enzymes responsible for these reactions are the cytochrome P-450 system. Carcinogenesis by polycyclic hydrocarbons has been attributed to the covalent modification of cellular macromolecules by these chemically unstable hydrocarbon metabolites (Conney, 1982). The formation of DNA adducts in particular has been considered to be a critical initiating event in this process (Brookes & Lawley, 1964; Singer, 1964). Recent investigations have demonstrated the existence of novel proteins that bind unmetabolized polycyclic aromatic hydrocarbons, and some polychlorinated compounds, in a reversible, noncovalent manner (Poland et al., 1976; Okey et al., 1979; Tierney et al., 1980; Zytkovicz, 1982; Collins & Marletta, 1984), and there has been increasing interest in the role these species may play in carcinogenesis. Binding affinities in the low nanomolar range and a finite number of sites per cell are the primary characteristics of these xenobiotic binding proteins. These features are reminiscent of hormone and drug receptor interactions (Burt, 1978). The TCDD' binding protein, or Ah receptor, from the C57B1/6J mouse is probably the best understood of these receptor-like proteins. Two distinct biological responses have been attributed to the Ah receptor. Its presence has been linked to the toxic manifestations of TCDD and chlorinated pesticide exposure in target tissues (Knutson & Poland, 1980, 1982), but the exact role of the receptor in this process is not yet understood. A second, better characterized function of the Ah receptor is the stimulation of microsomal drug-metabolizing activities. Independent investigations have shown that the Ah receptor participates in a mechanism of action analogous to steroid horSupported in part by USPHS Grant CA37770 awarded by DHHS, National Cancer Institute. This work is part of a Ph.D. thesis submitted to the Graduate Division of Massachusetts Institute of Technology. *Address correspondence to this author. Present address: Howard Hughes Medical Institute Research Laboratories, Departments of Medicine and Biochemistry, Duke University Medical Center, Duke University, Durham, NC 27710.
0006-2960/86/0425-4322$01.50/0
mone receptors. Following binding of a number of polycyclic aromatic and chlorinated hydrocarbons to the receptor, stimulation of new cytochrome P-450 mRNA and protein synthesis is accomplished by DNA binding and enhancement of the transcription of cytochrome P-450 genes (Greenlee & Poland, 1979; Tukey et al., 1982; Jones et al., 1985). Biochemical studies of the Ah receptor have identified a 9s sedimenting species in sucrose density gradients as a key component of the receptor (Okey et al., 1979; Hannah et al., 1981). Additional, more abundant binding species for polycyclic aromatic hydrocarbons have also been identified that are distinct from the Ah receptor. We (Collins & Marletta, 1984) and others (Holder et al., 1981; Zytkovicz, 1982) have described the ligand binding properties of these sites and some of their physical characteristics. It has been difficult to establish the biological significance of these binding sites, and it is tempting to speculate that some of these proteins may participate in the regulation of xenobiotic metabolism. A receptor role, similar to the Ah receptor, is one possibility. The existence of carrier proteins that may facilitate the metabolism of these lipophilic substrates has also been suggested, but supporting evidence remains inconclusive (Hanson-Painton et al., 1983). Our studies have been directed toward the purification of these binding proteins. We considered this approach to be a valuable one for addressing the issue of their biological function. Isolation of the binding protein is a prerequisite for the preparation of antibodies that can be used as histochemical probes and as specific inhibitors in a test of biological function. I Abbreviations: Ah, aryl hydrocarbon; I-AP-6B, I-aminopyreneSepharose 6B; B[a]P, benzo[a]pyrene; BSA, bovine serum albumin; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; GABA, y-aminobutyric acid; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; HEDG buffer, 25 mM Hepes, 5.0 mM EDTA, 1.0 mM dithiothreitol, and 10% glycerol, pH 7.6; kDa, kilodalton; 3-MC, 3methylcholanthrene; PMSF, phenylmethanesulfonyl fluoride; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin;TLC, thin-layer chromatography; UV, ultraviolet.
0 1986 American Chemical Society
BENZO[A]PYRENE B I N D I N G
PROTEIN
The primary structure of the protein and the architecture of the binding site may also be studied in greater detail. Information obtained in our earlier ligand binding studies has allowed us to develop purification strategies that are based on the affinity of these proteins for aromatic hydrocarbon ligands. The affinity chromatography resin 1-aminopyrene-Sepharose 6B was developed for this purpose (Collins et al., 1985). In this paper we describe the preparation and use of a photoaffinity label for these proteins, and their purification is presented. MATERIALS AND METHODS Animals. Male C57B1/6J mice, age 7 weeks, were obtained from Jackson Laboratories (Bar Harbor, ME) and were housed at 22 OC, 50% relative humidity, in a fluorescent light controlled room. Charles River Mouse Chow and distilled water were given ad libitum for 1 week, and then mice were killed by cervical dislocation at age 8 weeks. Chemicals. Reagent and specialty chemicals were obtained from the following sources: B[a]P, 1-aminopyrene, and 1nitropyrene from Aldrich Chemical Co. (Milwaukee, WI) (caution: these compounds have been determined to be carcinogenic or cancer-suspect agents; handling of these compounds was carried out in a fume hood with protective clothing and gloves; solutions containing these compounds were combined and disposed of as biohazardous waste); palladiumcoated polyethylenimine beads, epoxy-activated Sepharose 6B, and dextran from Sigma Chemical Co. (St. Louis, MO); DEAE-cellulose (DE52) from Whatman (Clifton, NJ); and Norit-A charcoal from Fisher Scientific (Pittsburgh, PA). Electrophoretic grade chemicals and SDS-polyacrylamide molecular weight standards (range 10 000-100 000) were obtained from Bio-Rad Laboratories (Richmond, CA). [3H]B[a]P (specific activity >78 Ci/mmol), 14C-methylated ovalbumin (M, 46 000), and 14C-methylatedBSA ( M , 69 000) were obtained from New England Nuclear (Boston, MA). [3H]B[a]Pwas repurified in two steps as previously described (Collins & Marletta, 1984). [3H]-1-Nitropyrene (23.7 Ci/ mmol) was a generous gift from Dr. John Groopman, Boston University (Boston, MA). Preparation of Liver Cytosol. Male C57B1/6J mice were sacrificed at 8 weeks of age, and hepatic cytosol was prepared as previously described (Collins & Marletta, 1984). HDG buffer (HEDG buffer without EDTA) containing 0.5 mM PMSF was used as the homogenization buffer for cytosol that was applied to DEAE-cellulose. Protein concentrations were assayed by the method of Bradford (1976) or by the fluorescamine method (Udenfriend et al., 1972) with crystalline BSA as the standard. Benzo[a]pyreneBinding Assay. Specific binding assays with [3H]B[a]P were performed as previously described (Collins & Marletta, 1984), except that the volume of the dextrancoated charcoal suspension was 0.25 mL. In some experiments using partially purified proteins the sample volume of the assay was 0.25 mL, with all other component volumes reduced accordingly. For saturation binding studies with the purified protein the assay was modified to contain 1 mg/mL BSA and 2-5 pg/mL purified protein. This adjustment was necessary in order to maintain a protein concentration in the assay of 1.O mg/mL. Binding incubations that contained 1-azidopyrene in either tritiated or nonradiolabeled form were performed at 10 OC for 2.5 h and were protected from light. Electrophoresis and Isoelectric Focusing. SDS-polyacrylamide slab gel electrophoresis was carried out by using a modification of the method of Laemmli (1970) as previously described (Collins & Marletta, 1984). Apparent molecular
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weights were determined from a semilogarithmic plot of the molecular weight of the standards (Bio-Rad, Richmond, CA) phosphorylase b (92 500), bovine serum albumin (66 200), ovalbumin (45 000), carbonic anhydrase (31 000), soybean trypsin inhibitor (21 500), and lysozyme (14 400) vs. migration distance. Gels were stained with silver (Morrissey, 1981). Isoelectric focusing was carried out as described in 3% polyacrylamide gels over a pH range of 4.5-7.1 (Tamkun & Hynes, 1983). Autoradiographs were prepared with Enlightening (New England Nuclear) and exposed to X-Omat AR film (Eastman Kodak Co., Rochester, NY). Synthesis of 1-Azidopyrene. The synthetic scheme for the preparation of 1-azidopyrene from 1-aminopyrene is essentially as described (Smith et al., 1981). Minor modifications of their procedure included doubling the molar equivalents of sodium nitrite and sodium azide, and the solvent system for silica gel chromatography was petroleum ether-methylene chloride (75:25). Purity was established by two-dimensional TLC (100% CHCl,, first dimension; 100% petroleum ether, second dimension) and comparison to reported UV spectrum and mass spectrum. Synthesis of [3H]-l-Azidopyrene.To a 0.30-mL Reacti-vial (Wheaton, Millville, NJ) set in an 85 OC water bath was added 100 pCi (3.38 nmol) of [3H]-l-nitropyrenedissolved in 200 pL of 95% (v/v) ethanol and 4 pL (82.3 nmol) of hydrazine hydrate. Addition of the catalyst (2.33 mg of palladium-coated polyethylenimine beads) was over a 3-h period. Progress of the reaction was monitored by TLC on 20-cm Bakerflex silica gel G F plates, which were developed in 100% CH2C12alongside authentic standards of 1-nitro-, 1-amino-, and 1-azidopyrene. The plates were cut into 1-cm strips and analyzed for tritium by scintillation counting. The reaction mixture from above was passed through a silanized Pasteur pipet containing crystals of MgSO4.7Hz0. The Reacti-vial and MgS04 were washed with a small volume of ethanol. The solvent was evaporated under a stream of nitrogen in a 0.30-mL Reacti-vial, and the [,HI- 1-aminopyrene was redissolved in 30 pL of acetone, 30 pL of H 2 0 , and 2.5 pL of 4 N HCl. The mixture was chilled on ice and sodium nitrite crystals were added in excess (0.78 mg, 14.7 pmol). The reaction was kept on ice for 1.5 h with brief mixing every 15 min, whereupon sodium azide (6.7 mg dissolved in 25 pL of H 2 0 ) was added by micropipet. This reaction was monitored by TLC, and the product mixture was passed through a small amount of MgS04 as described above. The [,H]-l-azidopyrene was purified by preparative silica gel TLC (1000-pm plates), eluted with CHZCl2,and stored at -20 "C. Photoaffinity Labeling by [ , H ] - 1 -Azidopyrene and Analysis of Labeled Proteins. Sample proteins (0.1-2.5 mg/mL as indicated in figure legends) were incubated, protected from light, with 100-200 nM [3H]-1-azidopyrene for 2.5 h on ice or were preincubated for 2 h with either 5 or 12.5 pM B[a]P, as indicated in figure legends, prior to the addition of [3H]-l-azidopyrene for 5 min. After brief treatment with dextran-coated charcoal to remove unbound ligand, samples were irradiated for 15-20 min at 300 nm in a Rayonet photochemical reactor (Southern New England Ultraviolet Co., Hamden, CT). The quartz reaction vessel was maintained at 4 "C by an internal water jacket. Some samples, due to their small volume (
